Capillary electrophoresis is a technique of considerable interest in the analysis of biological mixtures, particularly mixtures of small peptides proteins and nucleic acids, since it can be used on extremely small samples and permits the use of high voltages, thereby achieving separations at high speed.
One of the problems with capillary electrophoresis is the loading of the sample, i.e., its placement inside the end of the capillary in preparation for the separation. At present, this is commonly achieved by electrophoretic. electroendosmotic or pressure differential techniques.
In electrophoretic loading, a high voltage is used over a short period of time to transfer the sample from a sample reservoir into the capillary. The goal is to move small amounts of all species in the sample a short distance into the capillary. Once this is done, the sample reservoir is replaced with an appropriate buffer solution to permit one to proceed with electrophoretic separation of the loaded species.
What actually occurs in electroendosmotic loading is a combination of electrophoresis and electroendosmosis, with electroendosmosis having the predominating effect. The electroendosmosis varies from one experiment to the next, however, causing difficulties in the reproducibility of the sample volume.
Electroendosmosis can be suppressed by the application of appropriate coatings to the inside of the capillary tube, leaving electrophoresis as the sole driving force for the sample injection. Electrophoresis, however, has its own disadvantages. These arise from the differentials which necessarily exist among the various species in the sample in terms of their response to the electric potential. These differentials affect the distance which the species travel into the capillary during loading and thus the amounts of each species entering the capillary. Slowly migrating substances will thus migrate a shorter distance into the tube than will the faster migrating substances. Depending on the loading conditions, therefore, the composition of the applied sample may differ from that of the original sample.
Other variables in electrophoresis such as fluctuations in current strength may also enter into consideration. The significance and importance of these variables may vary.
In hydraulic loading, sample introduction is achieved by a pressure differential across the capillary either by applying a partial vacuum to the outlet end or a positive pressure to the inlet end. Problems with hydraulic loading arise from the limited extent to which one can control the pressure differential and its duration, two critical parameters which together govern the volume of the sample introduced.
It has now been discovered that bulk liquid flow may be achieved in a capillary tube in an electrophoretic system to a highly precise and reproducible degree, by imposing a controlled temperature change on the contents of either the tube or a closed vessel in fluid communication with the tube. The discovery is applicable to small volume introductions into the tube for purposes such as loading sample, as well as large volume transfers into or through the tube for purposes such as flushing the tube with buffer.
The temperature change may be a drop in temperature or a rise, and may occur between any initial and final temperature which will result in a controlled thermal contraction or expansion. Temperatures which do not involve a phase change of the heated or cooled medium, and thus bring about a continuous volume change will be preferred. In cases where the temperature change is imposed on the capillary tube itself, the temperatures will most conveniently be selected such that the final temperature is the temperature at which the separation is to be performed.
Thus, certain embodiments of the invention involve imposing a temperature change on the capillary itself. These embodiments are most useful in loading the capillary with a sample. In these embodiments, the capillary is filled with separation medium, and the temperature of the filled capillary is adjusted as necessary to prepare for a temperature drop of a magnitude calculated to achieve a selected volume contraction. The magnitude of the temperature drop is of course determined in conjunction with the length of capillary to which the temperature drop is applied, and this length may be either the entire capillary or a selected portion of its length.
Once the capillary temperature is equilibrated at the initial temperature, the end of the capillary in which the sample is to be introduced is submerged in a reservoir containing the sample. The temperature of the capillary or its temperature-controlled portion is then lowered to the final temperature, causing an aliquot of the sample is drawn into the capillary end. The final temperature is then maintained as the reservoir is replaced with an electrode buffer, and electrophoresis is performed. Appropriate and accurate selection of both the length of the capillary undergoing the temperature change and the magnitude of the temperature change will permit accurate and highly reproducible control of the size of the aliquot with no change in the proportion of the sample components as they enter the capillary.
In other embodiments of the invention, the temperature change is imposed on a bulb or other external vessel filled with liquid and joined to one end of the capillary in full fluid communication with the capillary contents. This permits the bulk fluid movement to occur without imposing any temperature change on the capillary itself. Depending on its location, the bulb may either push or pull liquid through the capillary, and may do so either directly or through intervening reservoirs. The temperature change may thus be either a rise or a drop. The use of a bulb also offers a wider range of volumetric flow since it is not limited to the dimensions of the capillary. When the volume of the bulb is large relative to the capillary volume sample loading may be achieved with a very small change in temperature. A bulb is particularly useful, however, in flushing the entire capillary with buffer.
Bulk fluid movement by either of these methods, whether it be loading of sample or flushing of the entire capillary, can be done with the same degree of precision as a thermometer. Further advantages, features and embodiments of the invention will be apparent from the description which follows.